NIR-II Excitation and NIR-I Emission Based Two-photon Fluorescence Lifetime Microscopic Imaging Using Aggregation-induced Emission Dots

Abstract

Near-infrared(NIR) lights are powerful tools to conduct deep-tissue imaging since NIR-I wavelengths hold less photon absorption and NIR-II wavelengths serve low photon scattering in the biological tissues compared with visible lights. Two-photon fluorescence lifetime microscopy(2PFLM) can utilize NIR-II excitation and NIR-I emission at the same time with the assistance of a well-designed fluorescent agent. Aggregation induced emission(AIE) dyes are famous for unique optical properties and could serve a large two-photon absorption(2PA) cross-section as aggregated dots. Herein, we report two-photon fluorescence lifetime microscopic imaging with NIR-II excitation and NIR-I emission using a novel deep-red AIE dye. The AIE-gens held a 2PA cross-section as large as 1.61×104 GM at 1040 nm. Prepared AIE dots had a two-photon fluorescence peak at 790 nm and a stable lifetime of 2.2 ns under the excitation of 1040 nm femtosecond laser. The brain vessels of a living mouse were vividly reconstructed with the two-photon fluorescence lifetime information obtained by our home-made 2PFLM system. Abundant vessels as small as 3.17 µm were still observed with a nice signal-background ratio at the depth of 750 µm. Our work will inspire more insight into the improvement of the working wavelength of fluorescent agents and traditional 2PFLM.